The allergic reaction in man and animals has been extensively studied and the basic immune mechanisms involved are well known. The generic name for molecules that cause an allergic reaction is allergen. There are numerous species of allergens. Common examples include plant pollens, bee venom, house dust, animal dander, and a wide array of food proteins. Many allergens are protein or polypeptide in nature, as proteins and polypeptides are generally more antigenic than carbohydrates or fats. The allergic reaction occurs when tissue-sensitizing immunoglobulin of the IgE type reacts with foreign allergen. The IgE antibody is bound to mast cells and/or basophils, and these specialized cells release chemical mediators of the allergic reaction when stimulated to do so by divalent antigens bridging the antibody molecules. Histamine, platelet activating factor, arachidonic acid metabolites, and serotonin are among the best known mediators of allergic reactions in man.
The symptoms of the allergic reaction vary, depending on the location within the body where the IgE reacts with the antigen. If the reaction occurs along the respiratory epithelium the symptoms are sneezing, coughing and asthmatic reactions. If the interaction occurs in the digestive tract, as in the case of food allergies, abdominal pain and diarrhea are common. Systematic reactions, for example following a bee sting, can be severe and often life threatening.
The preferred, but frequently impossible, method of relieving allergies is allergen avoidance. Failing that, there are two medical approaches to allergy control, both with an approximate 60 to 85% efficacy rate (Aas K., Allergy 37:1-14 (1982)). The most common approach to the medical treatment of allergies is to treat the symptom. Drugs known to block the effects of the chemical mediators of the allergic reactions, including antihistamines, are used to control the severity of the allergic symptoms. These drugs, however, do nothing to prevent the allergic reaction and the liberation of the chemical mediators, and do nothing to prevent or diminish allergic responses to subsequent allergen exposure.
Another approach is to prevent the allergic reaction by desensitizing the allergic host. This is accomplished by giving repeated small doses of the reactive allergen. The treatment usually involves injecting the allergens under the skin. Treatment with reactive allergens, more appropriately called immunotherapy, is believed to increase the concentration of antibodies of the IgG type against the allergen. The IgG antibody competes with the IgE antibody for allergen binding, and this competitive antibody somehow neutralizes, arrests, or blocks the action of the tissue sensitizing IgE antibody, although a distinct correlation between blocking antibody and amelioration of symptoms has not been definitely proven (Mailing, H. J., (ed.), Immunotherapy Position Paper, Allergy (Supp.) 6, 43:9-33 (1988)). This rationale, although generally accepted, is not fully understood, and does not reflect the complex interactions and events that accompany the IgG response. For example, other effects of immunotherapy that may be involved with relief of symptoms include suppression of IgE, increase in blocking IgA and IgG in secretions, reduced basophil reactivity/sensitivity, and reduced lymphocyte responsiveness to allergens. All of these changes may not occur in every patient, and those related to actual symptom relief have not been conclusively defined (Norman, P. S., J. Allergy Clin. Immunol. 75:531-545 (1985)).
Immunotherapy using reactive allergen is dangerous because the sensitized host is actually treated with the molecules capable of eliciting an allergic response. The treatment is started with extremely low does to avoid inducing a severe reaction. The antigen concentration required for 50% histamine release from peripheral basophils of an allergic individual varies 10,000 fold from patient to patient (Norman, P. S., J. Allergy Clin. Immunol. 75:531-545 (1985)). If no adverse reaction occurs, higher doses are given. The injection may cause severe allergic reactions and extreme care must be taken. Only experienced doctors can administer this treatment because if a severe reaction occurs immediate medical treatment must be given to control the symptoms of the allergic reaction. Desensitization is an expensive, painful, and time consuming process (Aas K., Allergy 37:1-14 (1982)), therefore, only the most severe allergies are treated by this method (Malling, H. J., ed., Immunotherapy Position Paper, Allergy (Supp.) 6, 43:9-33 (1988)).
Modifying allergens to eliminate their allergenicity, or ability to induce an IgE-mediated response, while preserving their immunogenicity, or their ability to elicit the protective IgG response, has been under investigation for years. Polymerized antigens have been evaluated with the theory that they would display reduced antigenicity due to concealed antigenic determinants, a lower molecular concentration hence decreased bridging opportunity on a weight basis, and slower diffusion through the tissues (Patterson R., J. Allergy Clin. Immunol. 68:85-90 (1981)). Allergen conjugates have been studied with a variety of allergens in attempts to selectively inhibit the formation of IgE antibodies while normal IgM and IgG responses to the antigen occurred (Lee, W. Y. et al., Imm. Rev. 41:200-217 (1978)). Conjugates with a glutamic acid/lysine copolymer may be acting specifically on IgE lymphocytes, on suppressor and/or helper T cells, or a combination of mechanisms may be in effect (Liu, F. T. et al., Proc. Natl. Acad. Sci. USA 76:1430-1434 (1979)).
Modifications that decrease the size of the allergen in efforts to eliminate their allergenicity have also been attempted. It has been proposed that limited proteolysis may actually reveal suppressor determinants on allergen molecules (Mowatt A. M., Immunology 56:253-260 (1985)).
Polypeptide fractions of allergens and the use of enzymes to break down the parent proteins is not new (King, T. P., Advances in Immunology 23:77 (1976)). U.S. Pat. No. 4,469,677 teaches the use of polypeptide fractions prepared from allergens for desensitization of allergic humans and animals. The polypeptide fractions are prepared by digesting allergens with proteolytic enzymes. Following the enzymatic digestion procedure, the enzymes and residual parent allergens must be removed from the polypeptide subfractions. The specific structure of the polypeptide fractions is dependent on the enzymes used for the digestive process. However, this is a synthetic process that requires pre-selection of the enzymes used for the digestive process. The polypeptide fraction is produced by controlled proteolytic digestion of the polypeptide allergen. Although this represents an improvement over using the reactive antigens, the treatment still requires frequent and painful injections.
The function of the polypeptide fractions for suppressing immune function are structure dependent. see Unanue, E. R. et al., Science 236:551-557 (1977) for a review of the structure dependence of polypeptide fractions of proteins in the immune response.
The structure of the polypeptide fractions of Michael is limited because the selection of enzymes is based on a limited understanding of which enzymes are most important.
The development of oral "tolerance" is a normal phenomenon, and a necessary function in response to the variety of foreign antigens consumed in the diet. Oral tolerance is initiated by a special class of T lymphocytes and their products, and it results in systematic suppression of the IgE-mediated hypersensitivity reaction. Variable responses of other components of the immune system have been reported to occur during induction of the tolerized state, so that the actual mechanism has not been succinctly defined.
In addition, animal studies have shown an actual enhancement of IgE production in response to intragastric administration of pollen extracts (Henderson, D. C. et al., Int. Archs. Allergy Appl. Immun. 79:66-71 (1986)). Although this may be a dose-related phenomenon, conflicting results were reported from two studies where 20 mg ovalbumin was administered orally to parenterally immunized mice, with IgE increased in one study (Handson, D. G. et al., Int. Arch. Allergy Appl. Immun. 55:256-532 (1977)) and decreased in the other (Lafont, S. et al., J. Exp. Ned. 155:1573-1578 (1982)).
Some investigators have concluded that recognition of antigenic determinants varies between parenterally and orally induced suppressor T cells. Suppressor cells resulting from feeding antigen were able to recognize different forms of the antigen, while parenterally induced suppressor T cells were specific for the molecular confirmation to which they initially responded (Mowatt, A. M., Immunology 56:253-260 (1985)). If this phenomenon is exhibited by a majority of antigens, it would further warrant the use of oral immunotherapy.
The problem with administering allergens orally to allergic subjects is that severe immune reactions may occur following treatment by this route as well. Anaphylaxis, relapse, urticaria, rectal bleeding, and anaphylactic shock have all been reported following oral immunotherapy (Platts-Mills, T. A. E., J. Allergy Clin. Immunol. 80:129-132 (1987)). In addition, oral treatment requires significantly larger doses than parenteral therapy, due to the action of the digestive tract enzymes upon the allergens. Individual responses to the ingested antigens may vary, however, so that more or less intact allergen may be represented to the gastrointestinal mucosa from the same dose given to different subjects, increasing the risk of inappropriate dosing.
Nonallergenic fractions of allergens can be given orally without concern for severe immune reactions; however, for reasons that are not clearly understood, oral administration of the polypeptide fractions of some allergens, including those prepared in the Michaels patent (U.S. 4,469,677) are not effective in causing desensitization. Studies of the immune response to feeding other modified proteins are limited, with varying results. Collagen-induced arthritis in mice was suppressed by feeding native type II collagen but not if the denatured molecule was fed (Nagler-Anderson, C. L. et al., Proc. Natl. Acad. Sci. USA 83:7443 (1986)). In a study of another autoimmune disease, both the disease-producing and nondisease-producing fragments and decapeptides of the myelin base protein were capable of eliciting immune suppression following oral treatment in rats (Higgins, P. J. et al., J. Immunology 140:440-445, (1988)). Results from a mouse study evaluating induction of oral tolerance with both natural and denatured ovalbumin identical suppression of delayed type hypersensitivity response to each form (Mowat, A. M., Immunology 56:253-260 (1985)).
One factor that may be involved in the failure of some molecular fragments to effectively induce immune tolerance is the low pH of the stomach, which may further modify the polypeptide fraction, abrogating its ability to induce tolerance.
Another factor involved in the failure of orally dosed allergen fragments to induce desensitization to future exposure may be the actual nature of the fragment itself. In vitro synthesis of fragments requires specific enzymes and conditions that may or may not result in the preparation of a biologically optimal formulation. The fragment may be effective when it is acutely exposed to the immune system following intravenous administration, but may be so susceptible to modification that any alterations occurring in the gastrointestinal tract before its exposure to the appropriate immune system components may destroy its effectiveness.
A need exists, therefore, for an oral formulation of allergen fractions that, when administered orally, is sufficiently active to induce tolerance but is not allergenic. A need also exists for methods of producing such products.